Xylitol is usually prepared by processes in which a xylan-containing material is first hydrolysed to produce a mixture of monosaccharides, including xylose. The xylose is then converted to xylitol, generally in a chemical process using a nickel catalyst such as Raney-nickel. A number of processes of this type have been described in the literature of the art. U.S. Pat. No. 3,784,408 (Jaffe et al.), U.S. Pat. No. 4,066,711 (Melaja et al.), U.S. Pat. No. 4,075,406 (Melaja et al.), U.S. Pat. No. 4,008,285 (Melaja et al.) and U.S. Pat. No. 3,586,537 (Steiner et al.) may be mentioned as examples.
Alternatively, xylitol can be produced from glucose. U.S. Pat. No. 5,096,820, Leleu et al., describes a process in which D-glucose is microbiologically converted to D-arabitol, which likewise is microbiologically converted to D-xylulose. The D-xylulose is then enzymatically isomerised into a mixture of D-xylose and D-xylulose, which is catalytically hydrogenated. Finally, the xylitol is recovered by chromatographic separation or crystallisation. U.S. Pat. No. 5,238,826, Leleu et al., uses a similar process to obtain xylose, ultimately for the preparation of xylitol by hydrogenation. The xylose is produced by microbiological conversion of D-glucose via D-arabitol to D-xylulose, which is then enzymatically isomerised into a mixture of D-xylose and D-xylulose. Finally, the mixture is subjected to chromatographic separation, the D-xylose fraction is recovered and the D-xylulose fraction is recirculated into the isomerisation step.
These prior methods are, however, technically complicated multistep processes which have relatively low efficiency. The greatest problems reside in achieving an effective and complete separation of xylose from other hydrolysis by-products. Thorough purification is essential, because the catalysts used in the reduction reaction of xylose are very sensitive. As regards the purity of the final product, the separation of xylitol from the other products produced in the reduction reaction will greatly depend on the amount of other components present. The more components, the more complicated and time-consuming purification and separation processes will be needed. Altogether, the production of xylitol by these processes is very costly.
Several attempts to utilise microorganisms for the biotechnological production of xylitol have also been reported. A main advantage of using microorganisms is that they are not as susceptible to varying reaction conditions as chemical catalysts. The production of xylitol by means of a biotechnological process is thus a highly attractive alternative, provided that such processes are able to provide a high quality product by a comparatively cost-effective method.
It is known that many yeast strains produce reductase enzymes that catalyse the reduction of sugars to corresponding sugar alcohols. Many yeasts, in particular Pichia, Candida, Hansenula and Kluyveromyces, are also capable of reducing xylose to xylitol as an initial step in their xylose metabolism, and several yeast strains are able to use xylose as a sole source of carbon and energy.
The reaction route or pathway of xylose utilisation for yeasts is in general the following: xylitol is synthesised in the first step by reduction of xylose to xylitol with the aid of xylose reductase. Xylitol is then metabolised by a series of successive steps. Xylitol is first oxidised to xylulose with xylitol dehydrogenase, xylulose is phosphorylated to xylulose-5-phosphate with xylulose kinase (also called xylulokinase), and then part of the xylulose-5-phosphate is converted to pyruvate via several intermediate steps. Also ethanol and CO2 can be formed. The relevant main products and by-products vary depending on the yeast strain and the fermentation conditions. The reactions are not tightly coupled, and consequently, some xylitol is often accumulated.
It has also been reported that some bacteria, such as Corynebacterium sp. and Enterobacter liquefaciens, produce xylitol by the same pathway, utilising xylose as the main metabolite. However, the yields obtained have been very poor. So far, only one species of filamentous fungus, Petromyces albertensis, has been shown to produce notable amounts of xylitol from xylose [Winkelhausen E. and Kuzmanova S., J. Ferment. Bioeng. 86:1-14 (1998)]. Hence, prior to the present invention, there has been no incentive to use bacteria or filamentous fungi for xylitol production.
Also as regards yeasts, the research has generally focused on attempts to identify yeast strains with enhanced ability to produce ethanol rather than xylitol. Nevertheless, xylitol production is relatively common among xylose-utilizing yeasts. For example, of 44 yeasts belonging to five different genera, Candida, Hansenula, Kluyveromyces, Pichia and Pachysolen, 42 produced some xylitol into the culture media [Barbosa. M. F. S. et al., J. Indust. Microbiol. 3241-3251 (1988) and Enzyme Microb. Technol. 10:66-81 (1988)].
It has been suggested to use such strains for the industrial production of xylitol. PCT publications WO 90/8193, WO 91/0740, WO 88/5467 and French published application 2 641 545 describe the use of Candida tropicalis, Candida guilliermondii and Candida parapsilosis, respectively.
U.S. Pat. No. 5,081,026, Heikkilä et al., describes a process for the production of xylitol from xylose, in which an aqueous xylose solution is fermented with a yeast strain capable of converting free xylose to xylitol and free hexoses to ethanol. After fermentation, a xylitol-rich fraction is obtained by chromatographic separation, and finally, xylitol is recovered from said fraction. In particular Candida and Debaryomyces are mentioned as suitable yeast species.
Profitable industrial production of xylitol by enzymatic bioconversion of xylose is possible only if the yield is high. No wild yeast strains have been shown to achieve this.
An attempt to solve this problem has been described in WO 91/15588, Hallborn, J. et al. The inventors cloned the xylose reductase gene from Pichia stipitis into Saccharomyces cerevisiae and obtained strains capable of converting xylose into xylitol with a claimed yield of 95% [Hallborn et al., Bio/Technology 9:1090 (1991)]. Saccharomyces cerevisiae does not normally express enzymes of the xylose pathway, but is widely accepted and commonly used in the food industry for other purposes, for example in bakery.
Gong C. et al., Biotechnol. Letters 3:125-130 (1981) describe two high xylitol producing yeast mutants denominated HXP 1 and HXP 2, obtained after UV-mutagenesis of a wild strain of Candida tropicalis which originally was capable of metabolising D-xylose into xylitol.
Mutants defective in xylose utilisation have also been described. Hagedorn J. et al., Curr. Genetic. 16:27-33 (1989) disclose mutants of the yeast Pichia stipitis which were unable to utilise xylose as the sole carbon source and which were deficient in either xylose reductase or xylitol dehydrogenase. Stevis, P. E. et al., Appl. Biochem. Biotechnol. 20:327-334 (1989) disclose the construction of yeast xylulokinase mutants by recombinant DNA techniques.
EP 0 604 429, Xyrofin, describes novel yeast strains with modified xylitol metabolism, a process for the production of said strains, and the use of said strains in a process for producing xylitol. The strains are capable of reducing xylose into xylitol, but are deficient in one or more enzymes involved in the xylitol metabolism, with the effect that the xylitol produced accumulates in the culture medium and can be recovered therefrom. The yeasts described belong to the genera Candida, Hansenula, Kluyveromyces or Pichia, and the genetic modification eliminates or reduces expression of the gene that encodes xylitol dehydrogenase or xylulose kinase, or both.
EP 0 672 161, Xyrofin, describes a method for the production of xylitol from carbon sources other than xylose and xylulose by using recombinant hosts. The microorganisms either produce xylitol via an altered arabitol route involving in particular arabitol dehydrogenase, or via altered (over)expression of genes encoding the enzymes of the oxidative branch of the pentose phosphate pathway (PPP), in particular glucose-6-phosphate dehydrogenase or 6-phospho-D-gluconate dehydrogenase, thus enabling utilisation of glucose, for instance. When used, D-glucose is phosphorylated into D-glucose-6-phosphate and converted to D-ribulose-5-phosphate via 6-phospho-D-gluconate. The D-ribulose-5-phosphate is then epimerised to D-xylulose-5-phosphate, dephosphorylated to D-xylulose and reduced to xylitol. Amplification of glucose-6-phosphate dehydrogenase enzyme activity in osmotolerant yeasts is also described in FR 2 772 788, Roquette Freres.
EP 0 974 646 A describes microorganisms capable of producing xylitol, or xylulose, from glucose. The microorganisms are naturally occurring and belong to the family Acetobacteracea. Novel strains of genus Asaia and Zucharibacter are mentioned as preferred.
Another approach that could be taken in the bioproduction of xylitol is the enhancement of xylose production, thus providing more xylose as the primary metabolite for xylitol production.
Some fungi, including Aureobasidium, Aspergillus, Trichoderma, Fusarium and Penicillium, have been reported to have xylanolytic activity and thus be able to degrade xylan-containing biopolymers and metabolise the xylan into xylose. In addition, several yeast species have been thoroughly studied, but their hydrolytic activity has not been the main target for the studies and has not been applied in large-scale industrial processes.
Kuhad R. C. et al., Process Biochemistry 33:641-647 (1998) describe a hyperxylanolytic mutant strain of Fusarium oxysporum produced by UV and N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment, and the enhancement of its xylanase production by optimisation of several nutritional and fermentation parameters, including temperature, pH, substrate and inoculum size.
Some Trichoderma strains have also been shown to be efficient producers of hemicellulases, in particular xylanase. The literature of the art also includes several reports on attempts to provide induction of β-xylosidase production during fermentation. Kristufek D. et al., Appl. Microbiol. Biotechnol. 42:713-717 (1995), using xylose, xylobiose and xylan as inducers for β-xylosidase induction in Trichoderma reesei, and Margolles-Clark E. et al., J. Biotechnol. 57:167-179 (1997), studying the expression of ten hemicellulase-encoding genes and using e.g. cellulose, xylobiose, xylan and L-arabitol as inducers, may be mentioned as examples.
The enhancement of hemicellulase activity by various gene manipulation techniques have also been reported. For instance Margolles-Clark E. et al., Appl. Environ. Microbiol., October 1996, 3840-3846, describe the isolation of the genes encoding β-xylosidase and α-L-arabinofuranosidase from Trichoderma reesei and cloning and expressing said genes in Saccharomyces cerevisiae. WO 97/00964, Rijksland-Bouwuniversiteit Wageningen, describes a novel β-xylosidase from Aspergillus niger, the nucleotide sequence encoding it and its use especially as a bread improver. Aspergillus, Trichoderma and Fusarium are mentioned as host cells. Hodits R. et al., ECB6: Proceedings of the 6th European Congress on Biotechnology, 13th to 17th June 1993, report on recombinant Trichoderma reesei strains producing improved and tailor-made xylanases, including a method of amplifying a gene encoding β-xylosidase. U.S. Pat. No. 5,837,515, Nevalainen et al., discloses a method for overproduction of hemicellulases, including endoxylanase, β-xylosidase, α-arabinosidase, α-D-glucuronidase and acetyl esterase, by cultivating Trichoderma reesei strains at least partially deficient in expressing cellulase enzyme(s) and transformed to include multiple copies of the gene encoding the desired enzyme(s).
Also bacteria have been studied in this respect. As an example may be mentioned Paice et al., Biotechnology and Bioengineering 32:235-239 (1988) describing in the article “Viscosity-enhancing bleaching of hardwood kraft pulp with xylanase from a cloned gene” Escherichia coli strains capable of overproduction of endoxylanase and β-xylosidase. For instance some Bacillus and Clostridium strains have also been reported to have xylanolytic activity.
The background art thus describes the production of xylitol from xylan by multistep chemical processes or by multistep combinations of chemical and biological processes. Further, processes utilising microorganisms, in particular yeasts, capable of producing xylitol from monosaccharide solutions or pure xylose solutions have been described. So far, no industrial processes for the production of xylitol from complex xylan-containing materials solely by means of a biotechnological method have been described, despite the fact that such a process would offer several advantages as compared to conventional processes, as far as efficient utilisation of the raw material and efficient production of xylitol in a cost-effective manner is concerned. There thus remains a constant need for further research and development in this area, with the aim of achieving efficient bioproduction of xylitol.